Free radicals play essential roles in many biochemical processes and are the key catalytic elements in free radical enzymes. These radical enzymes include the radical copper oxidases in which a stable tyrosine-cysteine dimer protein radical coupled to a copper ion form a metalloradical redox complex whose unique stability makes it an ideal model for investigating the role of protein free radicals in catalysis. Our goal is to define the role of the metalloradical complex in the catalytic mechanism of two radical copper oxidases (galactose oxidase and glyoxal oxidase), preparing alternative substrates for kinetic studies of substrate oxidation by both native and mutant active sites. The elementary catalytic steps will be probed using isotope kinetics, substrate profiling, and temperature perturbations. The role of quantum mechanical tunneling in radical catalysis by this class of enzymes will be systematically explored. We will also probe kinetic complexes formed during reoxidation of the enzyme by dioxygen, and explore the consequences of protein mutagenesis on the O2 reduction reaction. In addition to developing insight into the catalytic turnover mechanism, we will investigate the origin of the novel protein free radical redox site through cofactor biogenesis studies. This aspect of the project will take advantage of engineered yeast expression strains to produce unprocessed pre-protein for spectroscopic and kinetic analysis of maturation events. These experiments will also shed light on copper delivery to the pre-apoenzyme in the secretory pathway during export from the cell and in vivo formation of the active enzyme complex.